Modulators of the function of FAS/APO1 receptors

ABSTRACT

Proteins capable of modulating the function of FAS/APO1 are provided. The proteins may be prepared by culturing a host cell transformed with a vector containing the DNA encoding such a protein under suitable conditions and isolating the protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 08/860,082, filed Aug. 19, 1997, which is a §371 ofinternational application PCT/US95/16542, filed Dec. 14, 1995, theentire contents of each being hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is generally in the field of receptors belongingto the TNF/NGF superfamily of receptors and the control of theirbiological functions. The TNF/NGF superfamily of receptors includesreceptors such as the p55 and p75 tumor necrosis factor receptors(TNF-Rs) and the FAS ligand receptor (also called FAS/APO1 or FAS-R andhereinafter will be called FAS-R) and others. More specifically, thepresent invention concerns a novel protein, herein designated MORT-1(also called HF-1) which binds to the intracellular domain (IC) of theFas-R, (this intracellular domain designated Fas-IC) and which novelprotein is capable of modulating the function of the Fas-R. Further,MORT-I is also capable of self-association and can activate cellcytotoxicity on its own. The present invention also concerns thepreparation and uses of MORT-1.

It should be noted that HF-1 (the original designation) and MORT-1 (thepresently used designation) are both used throughout the specificationand denote the same protein.

BACKGROUND OF THE INVENTION AND PRIOR ART

Tumor Necrosis Factor (TNF-α) and Lymphotoxin (TNF-β(hereinafter, TNF,refers to both TNF-A and TNF-β) are multifunctional pro-inflammatorycytokines formed mainly by mononuclear phagocytes, which have manyeffects on cells (Wallach, D. (1986) in: Interferon 7 (Ion Gresser,ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami(1987)). Both TNF-α and TNF-β initiate their effects by binding tospecific cell surface receptors. Some of the effects are likely to bebeneficial to the organism: they may destroy, for example, tumor cellsor virus infected cells and augment antibacterial activities ofgranulocytes. In this way, TNF contributes to the defense of theorganism against tumors and infectious agents and contributes to therecovery from injury. Thus, TNF can be used as an anti-tumor agent inwhich application it binds to its receptors on the surface of tumorcells and thereby initiates the events leading to the death of the tumorcells. TNF can also be used as an anti-infectious agent.

However, both TNF-α and TNF-β also have deleterious effects. There isevidence that over-production of TNF-A can play a major pathogenic rolein several diseases. Thus, effects of TNF-α, primarily on thevasculature, are now known to be a major cause for symptoms of septicshock (Tracey et al., 1986). In some diseases, TNF may cause excessiveloss of weight (cachexia) by suppressing activities of adipocytes and bycausing anorexia, and TNF-A was thus called cachectin. It was alsodescribed as a mediator of the damage to tissues in rheumatic diseases(Beutler and Cerami, 1987) and as a major mediator of the damageobserved in graft-versus-host reactions (Piques et al., 1987). Inaddition, TNF is known to be involved in the process of inflammation andin many other diseases.

Two distinct, independently expressed, receptors, the p55 and p75TNF-As, which bind both TNF-α and TNF-β specifically, initiate and/ormediate the above noted biological effects of TNF. These two receptorshave structurally dissimilar intracellular domains suggesting that theysignal differently (See Hohmann et al., 1989; Engelmann et al., 1990;Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990;Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990).However, the cellular mechanisms, for example, the various proteins andpossibly other factors, which are involved in the intracellularsignaling of the p55 an p75 TNF-Rs have yet to be elucidated (InPCT/US95/05854 and as set forth also herein below, there are describedfor the first time, new proteins capable of binding to the p75IC andp55IC). It is this intracellular signaling, which occurs usually afterthe binding of the ligand, i.e., TNF (α or β, to the receptor, that isresponsible for the commencement of the cascade of reactions thatultimately result in the observed response of the cell to TNF.

As regards the above mentioned cytocidal effect of TNF, in most cellsstudied so far, this effect is triggered mainly by the p55 TNF-R.Antibodies against the extracellular domain (ligand binding domain) ofthe p55 TNF-R can themselves trigger the cytocidal effect (see EP412486) which correlates with the effectivity of receptor cross-linkingby the antibodies, believed to be the first step in the generation ofthe intracellular signaling process. Further, mutational studies(Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that thebiological function of the p55 TNF-R depends on the integrity of itsintracellular domain, and accordingly it has been suggested that theinitiation of intracellular signaling leading to the cytocidal effect ofTNF occurs as a consequence of the association of two or moreintracellular domains of the p55 TNF-R. Moreover, TNF (α and β) occursas a homotrimer and as such has been suggested to induce intracellularsignaling via the p55 TNF-R by way of its ability to bind to and tocross-link the receptor molecules, i.e., cause receptor aggregation. InPCT/US95/05854 and also hereinbelow there is described how the p55IC andp55DD can self-associate and induce, in a ligand-independent fashion,TNF-associated effects in cells.

Another member of the TNF/NGF superfamily of receptors is the FASreceptor (FAS-R) which has also been called the Fas antigen, acell-surface protein expressed in various tissues and sharing homologywith a number of cell-surface receptors including TNF-R and NGF-R. TheFAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991),and appears to serve as a negative selector of autoreactive T cells,i.e., during maturation of T cells, FAS-R mediates the apoptotic deathof T cells recognizing self-antigens. It has also been found thatmutations in the FAS-R gene (lpr) cause a lymphoproliferation disorderin mice that resembles the human autoimmune disease systemic lupuserythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for theFAS-R appears to be a cell-surface associated molecule carried by,amongst others, killer T cells (or cytotoxic T lymphocytes—CTLs), andhence when such CTLs contact cells carrying FAS-R, they are capable ofinducing apoptotic cell death of the FAS-R-carrying cells. Further, amonoclonal antibody has been prepared that is specific for FAS-R, thismonoclonal antibody being capable of inducing apoptotic cell death incells carrying FAS-R, including mouse cells transformed by cDNA encodinghuman FAS-R (Itoh et al., 1991).

It has also been found that various other normal cells, besides Tlymphocytes, express the FAS-R on their surface and can be killed by thetriggering of this receptor. Uncontrolled induction of such a killingprocess is suspected to contribute to tissue damage in certain diseases,for example, the destruction of liver cells in acute hepatitis.Accordingly, finding ways to restrain the cytotoxic activity of FAS-Rmay have therapeutic potential.

Conversely, since it has also been found that certain malignant cellsand HIV-infected cells carry the FAS-R on their surface, antibodiesagainst FAS-R, or the FAS-R ligand, may be used to trigger the FAS-Rmediated cytotoxic effects in these and thereby provide a means forcombating such malignant cells or HIV-infected cells (see Itoh et al.,1991). Finding yet other ways for enhancing the cytotoxic activity ofFAS-R may therefore also have therapeutic potential.

It has been a long felt need to provide a way for modulating thecellular response to TNF (α or β) and FAS-R ligand, for example, inpathological situations as mentioned above, where TNF or FAS-R ligand isover-expressed it is desirable to inhibit the TNF- or FAS-Rligand-induced cytocidal effects, while in other situations, e.g., woundhealing applications, it is desirable to enhance the TNF effect, or inthe case of FAS-R, in tumor cells or HIV-infected cells it is desirableto enhance the FAS-R mediated effect.

A number of approaches have been made by the present inventors (see forexample, European Application Nos. EP 186833, EP 308378, EP 398327 andEP 412486) to regulate the deleterious effects of TNF by inhibiting thebinding of TNF to its receptors using anti-TNF antibodies or by usingsoluble TNF receptors (being essentially the soluble extracellulardomains of the receptors) to compete with the binding of TNF to the cellsurface-bound TNF-Rs. Further, on the basis that TNF-binding to itsreceptors is required for the TNF-induced cellular effects, approachesby the present inventors (see for example IL 101769 and itscorresponding EP 568925) have been made to modulate the TNF effect bymodulating the activity of the TNF-Rs. Briefly, EP 568925 (IL 101769)relates to a method of modulating signal transduction and/or cleavage inTNF-Rs whereby peptides or other molecules may interact either with thereceptor itself or with effector proteins interacting with the receptor,thus modulating the normal functioning of the TNF-Rs. In EP 568925 thereis described the construction and characterization of various mutant p55TNF-Rs, having mutations in the extracellular, transmembranal, andintracellular domains of the p55 TNF-R. In this way regions within theabove domains of the p55 TNF-R were identified as being essential to thefunctioning of the receptor, i.e., the binding of the ligand (TNF) andthe subsequent signal transduction and intracellular signaling whichultimately results in the observed TNF-effect on the cells. Further,there is also described a number of approaches to isolate and identifyproteins, peptides or other factors which are capable of binding to thevarious regions in the above domains of the TNF-R, which proteins,peptides and other factors may be involved in regulating or modulatingthe activity of the TNF-R. A number of approaches for isolating andcloning the DNA sequences encoding such proteins and peptides; forconstructing expression vectors for the production of these proteins andpeptides; and for the preparation of antibodies or fragments thereofwhich interact with the TNF-R or with the above proteins and peptidesthat bind various regions of the TNF-R, are also set forth in EP 568925.However, EP 568925 does not specify the actual proteins and peptideswhich bind to the intracellular domains of the TNF-Rs (e.g., p55 TNF-R),nor does it describe the yeast two-hybrid approach to isolate andidentify such proteins or peptides which bind to the intracellulardomains of TNF-Rs. Similarly, heretofore there has been no disclosure ofproteins or peptides capable of binding the intracellular domain ofFAS-R.

Thus, when it is desired to inhibit the effect of TNF, or the FAS-Rligand, it would be desirable to decrease the amount or the activity ofTNF-Rs or FAS-R at the cell surface, while an increase in the amount orthe activity of TNF-Rs or FAS-R would be desired when an enhanced TNF orFAS-R ligand effect is sought. To this end the promoters of both the p55TNF-R and the p75 TNF-R have been sequenced, analyzed and a number ofkey sequence motifs have been found that are specific to varioustranscription regulating factors, and as such the expression of theseTNF-Rs can be controlled at their promoter level, i.e., inhibition oftranscription from the promoters for a decrease in the number ofreceptors, and an enhancement of transcription from the promoters for anincrease in the number of receptors (see IL 104355 and IL 109633).Corresponding studies concerning the control of FAS-R at the level ofthe promoter of the FAS-R gene have yet to be reported.

Further, it should also be mentioned that, while it is known that thetumor necrosis factor (TNF) receptors, and the structurally-relatedreceptor FAS-R, trigger in cells, upon stimulation by leukocyte-producedligands, destructive activities that lead to their own demise, themechanisms of this triggering are still little understood. Mutationalstudies indicate that in FAS-R and the p55 TNF receptor (p55-R)signaling for cytotoxicity involve distinct regions within theirintracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993;Itoh and Nagata, 1993). These regions (the ‘death domains’) havesequence similarity. The ‘death domains’ of both FAS-R and p55-R tend toself-associate. Their self-association apparently promotes that receptoraggregation which is necessary for initiation of signaling (seePCT/US95/05854, as well as Song et al., 1994; Wallach et al., 1994;Boldin et al., 1995) and at high levels of receptor expression canresult in triggering of ligand-independent signaling (PCT/US95/05854 andBoldin et al., 1995).

Thus, prior to PCT/US95/05854 and the present invention, there have notbeen provided proteins which may regulate the effect of ligandsbelonging to the TNF/NGF superfamily, such as the TNF or FAS-R ligandeffect on cells, by mediation of the intracellular signaling process,which signaling is probably governed to a large extent by theintracellular domains (ICs) of the receptors belonging to the TNF/NGFsuperfamily of receptors, such as those of the TNF-Rs, i.e., the p55 andp75 TNF-R intracellular domains (p55IC and p75IC, respectively), as wellas the FAS-IC.

Accordingly, it is one aim of the invention to provide proteins, beingMORT-1, analogs, fragments or derivatives thereof, which are capable ofbinding to the intracellular domain of the FAS-R, which proteins arepresently believed to be involved in the intracellular signaling processinitiated by the binding of FAS ligand to its receptor. The MORT-1protein, analogs, fragments and derivatives thereof of the presentinvention are distinct from the FAS-IC-binding proteins described in theearlier mentioned applications.

Another aim of the invention is to provide antagonists (e.g.,antibodies) to these FAS-IC binding molecules, being the MORT-1 protein,analogs fragments and derivatives, which may be used to inhibit thesignaling process, when desired, when such FAS-IC-binding proteins arepositive signal effectors (i.e., induce signaling), or to enhance thesignaling process, when desired, when such FAS-IC-binding proteins arenegative signal effectors (i.e., inhibit signaling).

Yet another aim of the invention is to use such MORT-1 protein, analogs,fragments and derivatives, to isolate and characterize additionalproteins or factors, which may, for example, be involved furtherdownstream in the signaling process, and/or to isolate and identifyother receptors further upstream in the signaling process to which theseMORT-1 protein, analogs, fragments and derivatives bind (e.g., otherFAS-Rs or related receptors), and hence, in whose function they are alsoinvolved. Moreover, it is an aim of the present invention to use theabove-mentioned MORT-1 protein, analogs, fragments and derivatives asantigens for the preparation of polyclonal and/or monoclonal antibodiesthereto. The antibodies, in turn, may be used, for example, for thepurification of the new MORT-1 protein from different sources, such ascell extracts or transformed cell lines.

Furthermore, these antibodies may be used for diagnostic purposes, e.g.,for identifying disorders related to abnormal functioning of cellulareffects mediated by the FAS-R receptor.

A further aim of the invention is to provide pharmaceutical compositionscomprising the above MORT-1 protein, analogs, fragments or derivatives,as well as pharmaceutical compositions comprising the above notedantibodies or other antagonists.

SUMMARY OF THE INVENTION

In accordance with the present invention, we have found a novel proteinwhich is capable of binding to the intracellular domain of the FAS-R.This FAS-IC-binding protein may act as a mediator or modulator of theFAS-R ligand effect on cells by way of mediating or modulating theintracellular signaling process which usually occurs following thebinding of the FAS-R ligand at the cell surface.

This novel protein has been designated HF1, or MORT-1 (for ‘Mediator ofReceptor Toxicity’), and in addition to its FAS-IC-binding specificityhas other characteristics (see Example 1), for example, it has a regionhomologous to the ‘death domain’ (DD) regions of the p55-TNF-R and FAS-R(p55-DD and FAS-DD), and thereby is also capable of self-association.MORT-1 is also capable of activating cell cytotoxicity on its own, anactivity possibly related to its self-association capability. It has nowalso been found that co-expression of the region in MORT-1 (HF1) thatcontains the ‘death domain’ homology sequence (MORT-DD, present in theC-terminal part of MORT-1) strongly interferes with FAS-induced celldeath, as would be expected from its ability to bind to the ‘deathdomain’ of the FAS-IC. Further, in the same experimental conditions itwas found that co-expression of the part of MORT-1 that does not containthe MORT-DD region (the N-terminal part of MORT-1, amino acids 1-117,‘MORT-1 head’) resulted in no interference of the FAS-induced cell deathand, if at all, a somewhat enhanced FAS-induced cell cytotoxicity.

Accordingly, the present invention provides a DNA sequence encoding aherein designated MORT-1 protein, analogs, or fragments thereof, all ofwhich are capable of binding to or interacting with the intracellulardomain of the FAS-ligand receptor (FAS-IC).

In particular, the present invention provides a DNA sequence selectedfrom the group consisting of:

(a) a cDNA sequence derived from the coding region of a native MORT-1protein;

(b) DNA sequences capable of hybridization to a cDNA of (a) undermoderately stringent conditions and which encode a biologically activeFAS-R intracellular domain-binding protein; and

(c) DNA sequences which are degenerate as a result of the genetic codeto the DNA sequences defined in (a) and (b) and which encode abiologically active FAS-R intracellular domain-binding protein.

A specific embodiment of the above DNA sequence of the invention is aDNA sequence encoding the protein MORT-1 comprising the sequencedepicted in FIG. 4.

The present invention also provides a MORT-1 protein, analogs, fragmentsor derivatives thereof encoded by any of the above sequences of theinvention, said proteins, analogs, fragments and derivatives beingcapable of binding to or interacting with the intracellular domain ofthe FAS-R.

A specific embodiment of the above protein of the invention is theMORT-1 protein having the deduced amino acid sequence depicted in FIG.4.

Also provided by the present invention are vectors encoding the aboveMORT-1 protein, analogs, fragments or derivatives of the invention,which contain the above DNA sequence of the invention, these vectorsbeing capable of being expressed in suitable eukaryotic or prokaryotichost cells; transformed eukaryotic or prokaryotic host cells containingsuch vectors; and a method for producing the MORT-1 protein, analogs,conditions suitable for the expression of said protein, analogs,fragments or derivatives, effecting post-translational modifications ofsaid protein as necessary for obtention of said protein and extractingsaid expressed protein, analogs, fragments or derivatives from theculture medium of said transformed cells or from cell extracts of saidtransformed cells.

In another aspect, the present invention also provides antibodies oractive derivatives or fragments thereof specific to the MORT-1 protein,analogs, fragments and derivatives thereof, of the invention.

By yet another aspect of the invention, there are provided various usesof the above DNA sequences or the proteins which they encode, accordingto the invention, which uses include amongst others:

(i) a method for the modulation of the FAS-R ligand effect on cellscarrying a FAS-R, comprising treating said cells with one or more MORT-1protein, analogs, fragments or derivatives, according to the invention,all of which being capable of binding to the intracellular domain andmodulating the activity of said FAS-R, wherein said treating of thecells comprises introducing into said cells said one or more MORT-lprotein, analogs, fragments or derivatives in a form suitable forintracellular administration or introducing into said cells, a DNAsequence encoding said one or more proteins, analogs, fragments orderivatives in the form of a suitable expression vector carrying saidsequence, said vector being capable of effecting the insertion of saidsequence into said cells in the way that said sequence is expressed insaid cells;

(ii) a method for modulating the FAS-R ligand effect on cells comprisingtreating said cells with MORT-1, analogs, fragments or derivativesthereof, all being capable of binding to the intracellular domain andmodifying the activity of FAS-R wherein said treating of cells comprisesintroducing into said cells said MORT-1, analogs, fragments orderivatives in a form suitable for intracellular introduction thereof,or introducing into said cells a DNA sequence encoding said MORT-1,analogs, fragments or derivatives in the form of a suitable vectorcarrying said sequence, said vector being capable of effecting theinsertion of said sequence into said cells in a way that said sequenceis expressed in said cells;

(iii) a method as in (ii) above wherein said treating of said cells isby transfection of said cells with a recombinant animal virus vectorcomprising the steps of:

(a) constructing a recombinant animal virus vector carrying a sequenceencoding a viral surface protein (ligand) that is capable of binding toa specific cell surface receptor on the surface of a FAS-R-carrying celland a second sequence encoding a protein selected from the MORT-1protein, analogs, fragments and derivatives of the invention, that whenexpressed in said cells is capable of modulating the activity of saidFAS-R; and

(b) infecting said cells with said vector of (a).

(iv) a method for modulating the FAS-R ligand effect on cells carrying aFAS-R comprising treating said cells with antibodies or activederivatives or fragments thereof according to the invention, saidtreating being by application of a suitable composition containing saidantibodies, active fragments or derivatives thereof to said cells,wherein when the MORT-1 proteins or portions thereof of said cells areexposed on the extracellular surface, said composition is formulated forextracellular application, and when said MORT-1 proteins areintracellular said composition is formulated for intracellularapplication;

(v) a method for modulating the FAS-R ligand effect on cells carrying aFAS-R comprising treating said cells with an oligonucleotide sequenceencoding an antisense sequence of at least part of the MORT-I sequenceof the invention, said oligonucleotide sequence being capable ofblocking the expression of the MORT-1 protein;

(vi) a method as in (v) above wherein said treating of cells is bytransfection of said cells with a recombinant animal virus vectorcomprising the steps of:

(a) constructing a recombinant animal virus vector carrying a sequenceencoding a viral surface protein (ligand) that is capable of binding toa specific cell surface receptor on the surface of a FAS-R-carrying celland a second sequence which is an oligonucleotide sequence encoding anantisense sequence of at least part of the MORT-1 sequence according tothe invention, said oligonucleotide sequence being capable of blockingthe expression of the MORT-1 protein when introduced into said cells bysaid virus; and

(b) infecting said cells with said vector of (a)

(vii) a method for treating tumor cells or HIV-infected cells, or otherdiseased cells, comprising:

(a) constructing a recombinant animal virus vector carrying a sequenceencoding a viral surface protein that is capable of binding to a tumorcell surface receptor or HIV-infected cell surface receptor or areceptor carried by other diseased cells and a sequence encoding aprotein selected from the MORT-1 protein, analogs, fragments andderivatives of the invention, that when expressed in said tumor,HIV-infected cell, or other diseased cell is capable of killing saidcell; and

(b) infecting said tumor or HIV-infected cells or other diseased cellswith said vector of (a).

(viii) a method for modulating the FAS-R ligand effect on cellscomprising applying the ribozyme procedure in which a vector encoding aribozyme sequence capable of interacting with a cellular mRNA sequenceencoding a MORT-1 protein of the invention is introduced into said cellsin a form that permits expression of said ribozyme sequence in saidcells, and wherein when said ribozyme sequence is expressed in saidcells it interacts with said cellular mRNA sequence and cleaves saidmRNA sequence resulting in the inhibition of expression of said MORT-1protein in said cells;

(ix) a method selected from any of the above methods wherein said MORT-1protein or said MORT-1 encoding sequence comprises at least that part ofthe MORT-1 protein which binds specifically to the FAS-IC, or at leastthat part of the MORT-1 encoding sequence that encodes that part of theMORT-1 protein which binds specifically to the FAS-IC;

(x) a method for isolating and identifying a protein capable of bindingto the intracellular domain of FAS-R comprising applying the procedureof non-stringent southern hybridization followed by PCR cloning, inwhich a sequence or parts thereof according to the invention is used asa probe to bind sequences from a cDNA or genomic DNA library, having atleast partial homology thereto, said bound sequences then amplified andcloned by the PCR procedure to yield clones encoding proteins having atleast partial homology to said sequences according to the invention.

The present invention also provides a pharmaceutical composition for themodulation of the FAS ligand—effect on cells comprising, as activeingredient, any one of the following: (i) a MORT-1 protein according tothe invention, its biologically active fragments, analogs, derivativesor mixtures thereof; (ii) a recombinant animal virus vector encoding aprotein capable of binding a cell surface receptor and encoding a MORT-1protein or its biologically active fragments or analogs according to theinvention; and (iii) an oligonucleotide sequence encoding an antisensesequence of the MORT-1 sequence of the invention, wherein saidoligonucleotide sequence may be the second sequence of the recombinantanimal virus vector of (ii) above.

It should be mentioned that MORT-1 has a distinct region which binds tothe FAS-IC and another distinct region which is involved inself-association of MORT-1, and accordingly, these distinct regions orparts thereof may be used independently to identify other proteins,receptors, etc. which are capable of binding to MORT-1 or to FAS-R andwhich may be involved in the MORT-1- or FAS-R- related intracellularsignaling processes. Further, MORT-1 may have other activitiesassociated with either of the above distinct regions or other regions ofMORT-1 or combinations thereof, for example, enzymatic activity, whichmay be related to the cell cytotoxic effects of MORT-1 on its own. Thus,MORT-1 may also be used to specifically identify other proteins,peptides, etc. which may be involved in such additional activitiesassociated with MORT-1.

Other aspects and embodiments of the present invention are also providedas arising from the following detailed description of the invention.

It should be noted that, where used throughout, the following terms:“Modulation of the FAS-ligand effect on cells”; and “Modulation of theMORT-1 effect on cells” are understood to encompass in vitro as well asin vivo treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B are reproductions of autoradiograms of SDS-PAGE gels (10%acrylamide) showing the interaction between HF1 (MORT-1) and FAS-IC invitro. FIG. 1A shows a control autoradiogram of an immunoprecipitate ofthe proteins (from extracts of HeLa cells transfected with the FLAG-HF1(FLAG-MORT1) fusion protein or with the luciferase cDNA (control), theimmunoprecipitation being performed with anti-FLAG antibody; and

FIG. 1B shows an autoradiogram of a representative gel performed toevaluate the in vitro interaction between HF1 and FAS-IC by way ofassessing, autoradiographically, the binding of[³⁵S]-methionine-metabolically labeled HF1 produced in transfected HeLacells as a fusion protein with the FLAG octapeptide (FLAG-MORT1) to GST,human and mouse GST-FAS-IC fusion protein (GST-huFAS-IC, GST-mFAS-IC)and GST-FAS-IC fusion proteins in which the FAS-IC contained an Ile toAla replacement mutation at position 225 (GST-mFAS-IC I225A). The [³⁵S]labeled proteins of the HeLa cells including the labeled FLAG-MORT1fusion protein having been first extracted were subjected to interactionwith the various GST and GST-FAS-IC proteins (bound to glutathionebeads) and then to SDS-PAGE. As controls in all of the interactionexperiments, extracts of HeLa cells transfected with luciferase weresubjected to interactions with the GST and GST-FAS-IC fusion proteinsand SDS-PAGE. FIGS. 1A and B are also described in Example 1.

FIGS. 2A, B and C are reproductions of autoradiograms of SDS-PAGE gels(10% acrylamide) on which were separated various immunoprecipitates fromtransfected HeLa cells and which show the in vivo interaction of HF1(MORT1) with FAS-IC. The HeLa cells were transfected with DNA constructsencoding: HF1-FLAG (FLAG-MORT1) fusion protein alone, HF1-FLAG fusionprotein and the human FAS-R (FLAG-MORT1+Fas/APO1) or human FAS-R alone(Fas/APO1) (FIG. 2A), or with HF1-FLAG fusion protein and the humanp55-R (FLAG-MORT1+p55−R) (FIG. 2B); or with HF1-FLAG fusion protein anda chimeric fusion protein between human FAS-R and p55-R in which theextracellular domain of the FAS-R web replaced with the correspondingregion of the p55-R (FLAG-MORT1+p55-FAS chimera) or the FAS-R-p55-Rchimeric fusion protein alone (p55-Fas chimera) (FIG. 2C). In all casesthe transfected cells were metabolically labeled with [³⁵S] cysteine (20μCi/ml) and [³⁵S]methionine (40 μCi/ml), and were subjected to proteinextraction. The protein extracts from the different transfected cellswere then immunoprecipitated with various antibodies being anti-FLAG,anti-FAS, anti-p75-R and anti-p55-R antibodies (αFLAG, βFAS, αβ75-R andαβ55-R in FIGS. 2A-C) and subjected to SDS-PAGE. On the left side ofFIG. 2A there is indicated the protein bands corresponding to FAS-R(Fas/APO1) and HF1-FLAG (FLAG-MORT1); between FIGS. 2A and B are shownthe relative positions of standard molecular weight markers (in kDa),and on the right hand side of FIG. 2C the protein bands corresponding top55R and the pS5-FAS chimera are indicated. FIGS. 2A-C are alsodescribed in Example 1.

FIG. 3 shows a reproduction of a Northern blot in which poly A+RNA (0.3μg) from HeLa cells transfected was probed with HF1 cDNA, as describedin Example 1.

FIGS. 4 depicts schematically the preliminary nucleotide (SEQ ID NO:1)and deduced amino acid sequence (SEQ ID NO:2) of HF1, as described inExample 1, in which the ‘death domain’ is underlined as is a possibletranslation start site, i.e., the underlined methionine residue atposition 49 (bold, underlined M). The asterisk indicates the translationstop codon (nucleotides 769-771). At the beginning and in the middle ofeach line are provided two numerals depicting the relative positions ofthe nucleotides and amino acids of the sequence with respect to thestart of the sequence (5′ end), in which the first numeral denotes thenucleotide and the second numeral denoted the amino acid.

FIG. 5 shows the results of experiments to determine the C-terminal endof MORT-1, wherein FIG. 5 is a reproduction of an autoradiogram of anSDS-PAGE gel (10% acrylamide) on which were separated variousMORT-1-FLAG fusion products expressed in HeLa cells and metabolicallylabeled with ³⁵S-cysteine and ³⁵S-methionine followed byimmunoprecipitation with either anti-FLAG monoclonal antibodies (M2)(lanes 2, 4 and 6) or as a control, anti-p75 TNF-R antibodies (49)(lanes 1, 3 and 5), as described in Example 1.

FIGS. 6 (A and B) depict graphically the ligand-independent triggeringof cytocidal effects in cells transfected with MORT-1, wherein cellviability was determined either by the neutral red uptake assay (FIG.6A), or for specifically determining the viability of cells thatexpressed the transfected DNA, by measuring the amounts of placentalalkaline phosphatase secreted into the medium (FIG. 6B). HeLa cells weretransfected with tetracycline-controlled expression vectors encoding HF1(MORT1), human FAS-IC, human p55-IC, or luciferase (control), and in allcases also with a cDNA encoding the secreted alkaline phosphatase, whichpermitted the evaluation of the effect of transient expression of theseproteins on the viability of the cells. In both FIGS. 6A and 6B the opengraphs represent transfected cells grown in the presence of tetracycline(1 μg/ml, to block expression) and the closed graphs representtransfected cells grown in the absence of tetracycline. FIGS. 6A and Bare also described in Example 1.

FIG. 7 is a graphic representation of the effects of different portionsof the MORT-1 protein on the cell cytotoxic effects mediated by FAS-R,as described in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one aspect, to a novel protein MORT-1(HF1) which is capable of binding to the intracellular domain of theFAS-R receptor, a member of the TNF/NGF superfamily and hence isconsidered as a mediator or modulator of FAS-R, having a role in, forexample, the signaling process that is initiated by the binding of FASligand to FAS-R. The amino acid and DNA sequences of MORT-1 according tothe invention also represent new sequences; they do not appear in the‘GENEBANK’ or ‘PROTEIN BANK’ data banks of DNA or amino acid sequences.

Thus, the present invention concerns the DNA sequence encoding theMORT-1 protein and the MORT-1 protein encoded by the DNA sequences.

Moreover, the present invention also concerns the DNA sequences encodingbiologically active analogs, fragments and derivatives of the MORT-1protein, and the analogs, fragments and derivatives encoded thereby. Thepreparation of such analogs, fragments and derivatives is by standardprocedure (see for example, Sambrook et al., 1989) in which in the DNAsequences encoding the MORT-1 protein, one or more codons may bedeleted, added or substituted by another, to yield analogs having atleast a one amino acid residue change with respect to the nativeprotein. Acceptable analogs are those which retain at least thecapability of binding to the intracellular domain of the FAS-R, or whichcan mediate any other binding or enzymatic activity, e.g., analogs whichbind the FAS-IC but which do not signal, i.e., do not bind to a furtherdownstream receptor, protein or other factor, or do not catalyze asignal-dependent reaction. In such a way analogs can be produced whichhave a so-called dominant-negative effect, namely, an analog which isdefective either in binding to the FAS-IC, or in subsequent signalingfollowing such binding. Such analogs can be used, for example, toinhibit the FAS-ligand-effect by competing with the naturalFAS-IC-binding proteins. Likewise, so-called dominant-positive analogsmay be produced which would serve to enhance the FAS ligand effect.These would have the same or better FAS-IC-binding properties and thesame or better signaling properties of the natural FAS-IC-bindingproteins. In an analogous fashion, biologically active fragments ofMORT-1 may be prepared as noted above with respect to the analogs ofMORT-1. Suitable fragments of MORT-1 are those which retain the FAS-ICbinding capability or which can mediate any other binding or enzymaticactivity as noted above. Accordingly, MORT-1 fragments can be preparedwhich have a dominant-negative or a dominant-positive effect as notedabove with respect to the analogs. It should be noted that thesefragments represent a special class of the analogs of the invention,namely, they are defined portions of MORT-1 derived from the full MORT-1sequence, each such portion or fragment having any of the above-noteddesired activities. Similarly, derivatives may be prepared by standardmodifications of the side groups of one or more amino acid residues ofthe MORT-1 protein, its analogs or fragments, or by conjugation of theMORT-1 protein, its analogs or fragments, to another molecule, e.g., anantibody, enzyme, receptor, etc., as are well known in the art.

The new MORT-1 protein, its analogs, fragments and derivatives have anumber of possible uses, for example:

(i) They may be used to mimic or enhance the function of FAS-R ligand,in situations where an enhanced FAS-R ligand effect is desired such asin anti-tumor, antiinflammatory or anti-HIV applications where the FAS-Rligand-induced cytotoxicity is desired. In this case the MORT-1 protein,its analogs, fragments or derivatives, which enhance the FAS-R ligandeffect, i.e., cytotoxic effect, may be introduced to the cells bystandard procedures known per se. For example, as the MORT-1 protein isintracellular and it should be introduced only into the cells where theFAS-R ligand effect is desired, a system for specific introduction ofthis protein into the cells is necessary. One way of doing this is bycreating a recombinant animal virus, e.g., one derived from Vaccinia, tothe DNA of which the following two genes will be introduced: the geneencoding a ligand that binds to cell surface proteins specificallyexpressed by the cells, e.g., ones such as the AIDS (HIV) virus gp120protein which binds specifically to some cells (CD4 lymphocytes andrelated leukemias) or any other ligand that binds specifically to cellscarrying a FAS-R, such that the recombinant virus vector will be capableof binding such FAS-R-carrying cells; and the gene encoding the MORT-1protein. Thus, expression of the cell-surface-binding protein on thesurface of the virus will target the virus specifically to the tumorcell or other FAS-R-carrying cell, following which the MORT-1 proteinencoding sequence will be introduced into the cells via the virus, andonce expressed in the cells will result in enhancement of the FAS-Rligand effect leading to the death of the tumor cells or otherFAS-R-carrying cells it is desired to kill. Construction of suchrecombinant animal virus is by standard procedures (see for example,Sambrook et al., 1989). Another possibility is to introduce thesequences of the MORT-1 protein in the form of oligonucleotides whichcan be absorbed by the cells and expressed therein. A furtherpossibility is by modifying the method described by Lin et al., inJournal of Biological Chemistry, Vol. 270, No. 24, pp. 14255-14258,1995.

(ii) They may be used to inhibit the FAS-R ligand effect, e.g., in casessuch as tissue damage in septic shock, graft-vs.-host rejection, oracute hepatitis, in which it is desired to block the FAS-R ligandinduced FAS-R intracellular signaling. In this situation it is possible,for example, to introduce into the cells, by standard procedures,oligonucleotides having the anti-sense coding sequence for the MORT-1protein, which would effectively block the translation of mRNAs encodingthe MORT-l protein and thereby block its expression and lead to theinhibition of the FAS-R ligand-effect. Such oligonucleotides may beintroduced into the cells using the above recombinant virus approach,the second sequence carried by the virus being the oligonucleotidesequence.

Another possibility is to use antibodies specific for the MORT-1 proteinto inhibit its intracellular signaling activity.

Yet another way of inhibiting the FAS-R ligand effect is by the recentlydeveloped ribozyme approach. Ribozymes are catalytic RNA molecules thatspecifically cleave RNAs. Ribozymes may be engineered to cleave targetRNAs of choice, e.g., the mRNAs encoding the MORT-1 protein of theinvention. Such ribozymes would have a sequence specific for the MORT-1mRNA and would be capable of interacting therewith (complementarybinding) followed by cleavage of the mRNA, resulting in a decrease (orcomplete loss) in the expression of the MORT-1 protein, the level ofdecreased expression being dependent upon the level of ribozymeexpression in the target cell. To introduce ribozymes into the cells ofchoice (e.g., those carrying FAS-R) any suitable vector may be used,e.g., plasmid, animal virus (retrovirus) vectors, that are usually usedfor this purpose (see also (i) above, where the virus has, as secondsequence, a cDNA encoding the ribozyme sequence of choice). (Forreviews, methods etc. concerning ribozymes see Chen et al., 1992; Zhaoand Pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayarnaet al., 1993; Cantor et al., 1993; Barinaga, 1993; Crisell et al., 1993and Koizumi et al., 1993).

(iii) They may be used to isolate, identify and clone other proteinswhich are capable of binding to them, e.g., other proteins involved inthe intracellular signaling process that are downstream of the TNF-R orFAS-R intracellular domain. For example, the MORT-1 protein, namely, theDNA sequence encoding it may be used in the yeast two-hybrid system (seeExample 1, below) in which the sequence of the MORT-1 protein will beused as “bait” to isolate, clone and identify from cDNA or genomic DNAlibraries other sequences (“preys”) encoding proteins which can bind tothe MORT-1 protein. In the same way, it may also be determined whetherthe MORT-1 protein of the present invention can bind to other cellularproteins, e.g., other receptors of the TNF/NGF superfamily of receptors.

(iv) The MORT-1 protein, its analogs, fragments or derivatives may alsobe used to isolate, identify and clone other proteins of the same class,i.e., those binding to FAS-R intracellular domain or to functionallyrelated receptors, and involved in the intracellular signaling process.In this application the above noted yeast two-hybrid system may be used,or there may be used a recently developed system employing non-stringentsouthern hybridization followed by PCR cloning (Wilks et al., 1989). Inthe Wilks et al. publication, there is described the identification andcloning of two putative protein-tyrosine kinases by application ofnon-stringent southern hybridization followed by cloning by PCR based onthe known sequence of the kinase motif, a conceived kinase sequence.This approach may be used, in accordance with the present inventionusing the sequence of the MORT-1 protein to identify and clone those ofrelated FAS-R intracellular domain-binding proteins.

(v) Yet another approach to utilizing the MORT-1 protein, its analogs,fragments or derivatives of the invention is to use them in methods ofaffinity chromatography to isolate and identify other proteins orfactors to which they are capable of binding, e.g., other receptorsrelated to FAS-R or other proteins or factors involved in theintracellular signaling process. In this application, the MORT-1protein, its analogs, fragments or derivatives of the present invention,may be individually attached to affinity chromatography matrices andthen brought into contact with cell extracts or isolated proteins orfactors suspected of being involved in the intracellular signalingprocess. Following the affinity chromatography procedure, the otherproteins or factors which bind to the MORT-1 protein, its analogs,fragments or derivatives of the invention, can be eluted, isolated andcharacterized.

(vi) As noted above, the MORT-1 protein, its analogs, fragments orderivatives of the invention may also be used as immunogens (antigens)to produce specific antibodies thereto. These antibodies may also beused for the purposes of purification of the MORT-1 protein either fromcell extracts or from transformed cell lines producing MORT-1, itsanalogs or fragments. Further, these antibodies may be used fordiagnostic purposes for identifying disorders related to abnormalfunctioning of the FAS-R ligand system, e.g., overactive or underactiveFAS-R ligand-induced cellular effects. Thus, should such disorders berelated to a malfunctioning intracellular signaling system involving theMORT-1 protein, such antibodies would serve as an important diagnostictool.

(vii) MORT-1 may also be used as an indirect modulator of a number ofother proteins by virtue of its capability of binding to otherintracellular proteins, (the so-called MORT-1 binding proteins, seebelow), which other intracellular proteins directly bind yet otherintracellular proteins or an intracellular domain of a transmembraneprotein. An example of such a protein or such an intracellular domain isthe well-known p55 TNF receptor, the intracellular signaling of which ismodulated by a number of proteins which bind directly to itsintracellular domain (see copending IL 109632). In fact we have isolatedsuch a MORT-1 binding protein (see below and Example 2) which binds tothe intracellular domain of the p55 TNF receptor.

For the purposes of modulating these other intracellular proteins or theintracellular domains of transmembranal proteins, MORT-1 may beintroduced into cells in a number of ways as mentioned hereinabove in(ii).

It should also be noted that the isolation, identification andcharacterization of the MORT-1 protein of the invention may be performedusing any of the well known standard screening procedures. For example,one of these screening procedures, the yeast two-hybrid procedure as isset forth herein (Example 1), was used to identify the MORT-1 protein ofthe invention. Likewise as noted above and below, other procedures maybe employed such as affinity chromatography, DNA hybridizationprocedures, etc. as are well known in the art, to isolate, identify andcharacterize the MORT-1 protein of the invention or to isolate, identifyand characterize additional proteins, factors, receptors, etc. which arecapable of binding to the MORT-1 protein of the invention.

Furthermore, it should also be noted that amongst the characteristics ofMORT-1 is its ability to bind to the FAS-IC and also its ability toself-associate. MORT-1 is also capable of activating cell cytotoxicityon its own, an activity related to its self-association ability. Itappears (see Example 1) that the part of MORT-1 which binds to theFAS-IC is distinct from the part of MORT-1 which is involved in itsself-association. MORT-1 may also have other activities which may-be afunction of the above noted distinct parts of the MORT-1 molecule orother parts of the molecule or combinations of any of these parts. Theseother activities may be enzymatic or related to binding other proteins(e.g., MORT-1-binding proteins or other receptors, factors, etc.). Thus,MORT-1 may be used in the above methods for modulation of FAS-R-ligandeffects or its own MORT-1-mediated cellular effects, or it may be usedin the modulation of other cellular signaling processes related to otherreceptors, factors, etc.

More specifically, by this aspect of the invention the MORT-1 encodingDNA molecule itself and mutations thereof (i.e., encoding analogs oractive fractions of MORT-1) can be used for gene therapy (i.e., by theways set forth in uses (i) and (ii) above) for modulating the activityof the FAS-R (or modulating or mediating the FAS ligand-effect oncells). Moreover, as MORT-1 also has a cytotoxic effect on cells, theseMORT-1 or mutant MORT-1 encoding DNA molecules may also be used for genetherapy for modulating the MORT-1 effect in cells (also by way of theuses (i) and (ii) above). In these gene therapy applications, theMORT-1, analogs or derivatives may be used in three ways:

(a) the whole MORT-1 protein, its analogs, derivatives or activefragments which have both FAS-IC and ORT-1-binding ability (i.e.,contain the two regions of MORT-1, one of which is involved in bindingto FAS-IC and the other which is involved in the self-association ofMORT-1) may be used to modulate FAS-R and MORT-1-associated effects;

(b) the part of MORT-1, and analogs, derivatives, and active fragmentsof this part which binds to the FAS-IC may be used for inducing a‘dominant negative’ effect on FAS-IC, i.e., inhibition of FAS-R-mediatedcellular effects, or may be used for inducing a ‘gain of function’effect on FAS-IC, i.e., enhancement of the FAS-R-mediated cellulareffects; and

(c) the part of MORT-1 and analogs, derivatives and active fractions ofthis part, which binds specifically to MORT-1 may be used for inductionof ‘dominant negative’ or ‘gain of function’ effects on MORT-1, i.e.,either inhibition or enhancement of MORT-1-associated cellular effects.

As set forth in use (vi) above, the MORT-1 protein may be used togenerate antibodies specific to MORT-1. These antibodies or fragmentsthereof may be used as set forth hereinbelow in detail, it beingunderstood that in these applications the antibodies or fragmentsthereof are those specific for MORT-1.

As regards the antibodies mentioned herein throughout, the term“antibody” is meant to include polyclonal antibodies, monoclonalantibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id)antibodies to antibodies that can be labeled in soluble or bound form,as well as fragments thereof provided by any known technique, such as,but not limited to enzymatic cleavage, peptide synthesis or recombinanttechniques.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which populations containssubstantially similar epitope binding sites. MAbs may be obtained bymethods known to those skilled in the art. See, for example Kohler andMilstein, Nature 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubel etal., eds., Harlow and Lane ANTIBODES: A LABORATORY MANUAL, Cold SpringHarbor Laboratory (1988); and Colligan et al., eds., Current Protocolsin Immunology, Greene publishing Assoc. and Wiley Interscience N.Y.,(1992, 1993), the contents of which references are incorporated entirelyherein by reference. Such antibodies may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridomaproducing a mAb of the present invention may be cultivated in vitro, insitu or in vivo. Production of high titers of mAbs in vivo or in situmakes this the presently preferred method of production.

Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having the variableregion derived from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityin application and to increase yields in production, for example, wheremurine mAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mAbs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al., Proc. Natl. Acad. Sci. USA 81:32733277 (1984); Morrisonet al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne etal., Nature 312:643-646 (1984); Cabilly et al., European PatentApplication 125023.

(published Nov. 14, 1984); Neuberger et al., Nature 314:268-270 (1985);Taniguchi et al., European Patent Application 171496 (published Feb. 19,1985); Morrison et al., European Patent Application 173494 (publishedMarch 5, 1986); Neuberger et al., PCT Application WO 8601533, (publishedMarch 13, 1986); Kudo et al., European Patent Application 184187(published June 11, 1986); Sahagan et al., J. Immunol. 137:1066-1074(1986); Robinson et al., International Patent Application No. WO8702671(published May 7, 1987); Liu et al., Proc. Natl. Acad Sci USA84:3439-3443 (1987); Sun et al., Proc. Natl. Acad Sci USA 84:214-218(1987); Better et al., Science 240:1041-1043 (1988); and Harlow andLane, ANTIBODIES:A LABORATORY MANUAL, supra. These references areentirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the mAb with the mAb to which an anti-Id is being prepared. Theimmunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-Id antibody). See, for example,U.S. Pat. No. 4,699,880, which is herein entirely incorporated byreference.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

Accordingly, mAbs generated against the MORT-1 protein, analogs,fragments or derivatives thereof, of the present invention may be usedto induce anti-Id antibodies in suitable animals, such as BALB/c mice.Spleen cells from such immunized mice are used to produce anti-Idhybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can becoupled to a carrier such as keyhole limpet hemocyanin (KLH) and used toimmunize additional BALB/c mice. Sera from these mice will containanti-anti-Id antibodies that have the binding properties of the originalmAb specific for an epitope of the above MORT-1 protein, analogs,fragments or derivatives.

The anti-Id mAbs thus have their own idiotypic epitopes, or “idiotopes”structurally similar to the epitope being evaluated, such as GRBprotein-α.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F(ab′)₂, whichare capable of binding antigen. Fab and F(ab′)₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab′) ₂ and other fragments of theantibodies useful in the present invention may be used for the detectionand quantitation of the MORT-1 protein according to the methodsdisclosed herein for intact antibody molecules. Such fragments aretypically produced by proteolytic cleavage, using enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments).

An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The antibodies, including fragments of antibodies, useful in the presentinvention may be used to quantitatively or qualitatively detect theMORT-1 in a sample or to detect presence of cells which express theMORT-1 of the present invention. This can be accomplished byimmunofluorescence techniques employing a fluorescently labeled antibody(see below) coupled with light microscopic, flow cytometric, orfluorometric detection.

The antibodies (or fragments thereof) useful in the present inventionmay be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of the MORT-1 of thepresent invention. In situ detection may be accomplished by removing ahistological specimen from a patient, and providing the labeled antibodyof the present invention to such a specimen. The antibody (or fragment)is preferably provided by applying or by overlaying the labeled antibody(or fragment) to a biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of theMORT-1, but also its distribution on the examined tissue. Using thepresent invention, those of ordinary skill will readily perceive thatany of wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve such in situ detection.

Such assays for the MORT-1 of the present invention typically comprisesincubating a biological sample, such as a biological fluid, a tissueextract, freshly harvested cells such as lymphocytes or leukocytes, orcells which have been incubated in tissue culture, in the presence of adetectably labeled antibody capably of identifying the MORT-1 protein,and detecting the antibody by any of a number of techniques well knownin the art.

The biological sample may be treated with a solid phase support orcarrier such as nitrocellulose, or other solid support or carrier whichis capable of immobilizing cells, cell particles or soluble proteins.The support or carrier may then be washed with suitable buffers followedby treatment with a detectably labeled antibody in accordance with thepresent invention, as noted above. The solid phase support or carriermay then be washed with the buffer a second time to remove unboundantibody. The amount of bound label on said solid support or carrier maythen be detected by conventional means.

By “solid phase support”, “solid phase carrier”, “solid support”, “solidcarrier”, “support” or “carrier” is intended any support or carriercapable of binding antigen or antibodies. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon amylases, natural and modified celluloses,polyacrylamides, gabbros and magnetite. The nature of the carrier can beeither soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports or carriers include polystyrene beads. Those skilledin the art will know may other suitable carriers for binding antibody orantigen, or will be able to ascertain the same by use of routineexperimentation.

The binding activity of a given lot of antibody, of the invention asnoted above, may be determined according to well-known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

One of the ways in which an antibody in accordance with the presentinvention can be detectably labeled is by linking the same to an enzymeand use in an enzyme immunoassay (EIA). This enzyme, in turn, when laterexposed to an appropriate substrate, will react with the substrate insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymeswhich can be used detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactivity labeling the antibodies orantibody fragments, it is possible to detect R-PTPase through the use ofa radioimmunoassay (RIA). A good description of RIA may be found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company, NY (1978) with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard, T, incorporated by reference herein.The radioactive isotope can be detected by such means as the use of a γcounter or a scintillation counter or by autoradiography.

It is also possible to label an antibody in accordance with the presentinvention with a fluorescent compound. When the fluorescently labeledantibody is exposed to light of the proper wavelength, its presence canbe then detected due to fluorescence. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as 152E, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted forutilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the antibody bound to the solid phase is first contacted with thesample being tested to extract the antigen from the sample by formationof a binary solid phase antibody-antigen complex. After a suitableincubation period, the solid support or carrier is washed to remove theresidue of the fluid sample, including unreacted antigen, if any, andthe contacted with the solution containing an unknown quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support or carrier through the unlabeledantibody, the solid support or carrier is washed a second time to removethe unreacted labeled antibody.

In another type of “sandwich” assay, which may also be useful with theantigens of the present invention, the so-called “simultaneous” and“reverse” assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support or carrierand labeled antibody are both added to the sample being tested at thesame time. After the incubation is completed, the solid support orcarrier is washed to remove the residue of fluid sample and uncomplexedlabeled antibody. The presence of labeled antibody associated with thesolid support or carrier is then determined as it would be in aconventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support or carrier after a suitable incubationperiod is utilized. After a second incubation, the solid phase is washedin conventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support or carrier is thendetermined as in the “simultaneous” and “forward” assays.

The MORT-1 of the invention may then be produced by any standardrecombinant DNA procedure (see for example, Sambrook, et al., 1989) inwhich suitable eukaryotic or prokaryotic host cells are transformed byappropriate eukaryotic or prokaryotic vectors containing the sequencesencoding for the proteins. Accordingly, the present invention alsoconcerns such expression vectors and transformed hosts for theproduction of the proteins of the invention. As mentioned above, theseproteins also include their biologically active analogs, fragments andderivatives, and thus the vectors encoding them also include vectorsencoding analogs and fragments of these proteins, and the transformedhosts include those producing such analogs and fragments. Thederivatives of these proteins are the derivatives produced by standardmodification of the proteins or their analogs or fragments, produced bythe transformed hosts.

The present invention also relates to pharmaceutical compositionscomprising recombinant animal virus vectors encoding the MORT-1 protein,which vector also encodes a virus surface protein capable of bindingspecific target cell (e.g., cancer cells) surface proteins to direct theinsertion of the MORT-1 sequence into the ceils. Other aspects of theinvention will be apparent from the following examples.

The invention will now be described in more detail in the followingnon-limiting examples and the accompanying drawings:

EXAMPLE 1 Cloning and Isolation of the MORT-1 Protein Which Binds to theIntracellular Domain of the FAS-R

(i) Two-Hybrid Screen and Two-Hybrid β-Galactosidase Expression Test

To isolate proteins interacting with the intracellular domain of theFAS-R, the yeast two-hybrid system was used (Fields and Song, 1989; seealso co-pending IL 109632, 112002 and 112692). Briefly, this two-hybridsystem is a yeast-based genetic assay to detect specific protein-proteininteractions in vivo by restoration of a eukaryotic transcriptionalactivator such as GAL4 that has two separate domains, a DNA binding andan activation domain, which domains when expressed and bound together toform a restored GAL4 protein, is capable of binding to an upstreamactivating sequence which in turn activates a promoter that controls theexpression of a reporter gene, such as lacZ or HIS3, the expression ofwhich is readily observed in the cultured cells. In this system thegenes for the candidate interacting proteins are cloned into separateexpression vectors. In one expression vector the sequence of the onecandidate protein is cloned in phase with the sequence of the GAL4DNA-binding domain to generate a hybrid protein with the GAL4DNA-binding domain, and in the other vector the sequence of the secondcandidate protein is cloned in phase with the sequence of the GAL4activation domain to generate a hybrid protein with the GAL4-activationdomain. The two hybrid vectors are then co-transformed into a yeast hoststrain having a lacZ or HIS3 reporter gene under the control of upstreamGAL4 binding sites. Only those transformed host cells (co-transformants)in which the two hybrid proteins are expressed and are capable ofinteracting with each other, will be capable of expression of thereporter gene. In the case of the lacZ reporter gene, host cellsexpressing this gene will become blue in color when X-gal is added tothe cultures. Hence, blue colonies are indicative of the fact that thetwo cloned candidate proteins are capable of interacting with eachother.

Using this two-hybrid system, the intracellular domain, FAS-IC, wascloned, separately, into the vector pGBT9 (carrying the GAL4 DNA-bindingsequence, provided by Clontech, USA, see below), to create fusionproteins with the GAL4 DNA-binding domain. For the cloning of FAS-R intopGBT9, a clone encoding the full-length cDNA sequence of FAS-R (seeco-pending IL 111125) was used from which the intracellular domain (IC)was excised by standard procedures using various restriction enzymes andthen isolated by standard procedures and inserted into the pGBT9 vectoropened, in its multiple cloning site region (MCS), with thecorresponding suitable restriction enzymes. It should be noted that theFAS-IC extends from residues 175-319 of the intact FAS-R, this portioncontaining residues 175-319 being the FAS-IC inserted into the pGBT9vector (see also IL 111125).

The above hybrid (chimeric) vector was then co-transfected together witha cDNA library from human HeLa cells cloned into the pGAD GH vector,bearing the GAL4 activating domain, into the HF7c yeast host strain (allthe above-noted vectors, pGBT9 and pGAD GH carrying the HeLa cell cDNAlibrary, and the yeast strain were purchased from Clontech Laboratories,Inc., USA, as a part of MATCHMAKER Two-Hybrid System, #PT1265-1). Theco-transfected yeasts were selected for their ability to grow in mediumlacking Histidine (His⁻ medium), growing colonies being indicative ofpositive transformants. The selected yeast clones were then tested fortheir ability to express the lacZ gene, i.e., for their LAC Z activity,and this by adding X-gal to the culture medium, which is catabolized toform a blue colored product by β-galactosidase, the enzyme encoded bythe lacZ gene. Thus, blue colonies are indicative of an active lacZgene. For activity of the lacZ gene, it is necessary that the GAL4transcription activator be present in an active form in the transformedclones, namely that the GAL4 DNA-binding domain encoded by the abovehybrid vector be combined properly with the GAL4 activation domainencoded by the other hybrid vector. Such a combination is only possibleif the two proteins fused to each of the GAL4 domains are capable ofstably interacting (binding) to each other. Thus, the His⁺ and blue (LACZ⁺) colonies that were isolated are colonies which have beenco-transfected with a vector encoding FAS-IC and a vector encoding aprotein product of human HeLa cell origin that is capable of bindingstably to FAS-IC.

The plasmid DNA from the above His⁺, LAC Z⁺ yeast colonies was isolatedand electroporated into E. coli strain HB101 by standard proceduresfollowed by selection of Leu+and Ampicillin resistant transformants,these transformants being the ones carrying the hybrid pGAD GH vectorwhich has both the Amp^(R) and Leu² coding sequences. Such transformantstherefore are clones carrying the sequences encoding newly identifiedproteins capable of binding to the FAS-IC. Plasmid DNA was then isolatedfrom these transformed E. coli and retested by:

(a) retransforming them with the original FAS-R intracellular domainhybrid plasmid (hybrid pGTB9 carrying the FAS-IC) into yeast strain HF7as set forth hereinabove. As controls, vectors carrying irrelevantprotein encoding sequences, e.g., pACT-lamin or pGBT9 alone were usedfor co-transformation with the FAS-IC-binding protein (i.e.,MORT-1)encoding plasmid. The co-transformed yeasts were then tested forgrowth on His⁻ medium alone, or with different levels of3-aminotriazole; and

(b) retransforming the plasmid DNA and original FAS-IC hybrid plasmidand control plasmids described in (a) into yeast host cells of strainSFY526 and determining the LAC Z⁺ activity (effectivity of β-galformation, i.e., blue color formation).

The results of the above tests revealed that the pattern of growth ofcolonies in His⁻ medium was identical to the pattern of LAC Z activity,as assessed by the color of the colony, i.e., His colonies were also LACZ⁺. Further, the LAC Z activity in liquid culture (preferred cultureconditions) was assessed after transfection of the GAL4 DNA-binding andactivation-domain hybrids into the SFY526 yeast hosts which have abetter LAC Z inducibility with the GAL4 transcription activator thanthat of the HF-7 yeast host cells.

Using the above procedure a protein called HF1, and now also calledMORT-1 for “Mediator of Receptor-induced Toxicity”, was identified,isolated and characterized.

Furthermore, it should also be mentioned that in a number of the abovetwo-hybrid, β-galactosidase expression tests, the expression ofβ-galactosidase was also assessed by a preferred filter assay. In thescreening, 5 of about 3×10⁶ cDNAs were found to contain the MORT-1insert. The so-isolated cloned MORT-1 cDNA inserts were then sequencedusing standard DNA sequencing procedures. The amino acid sequence ofMORT-1 was deduced from the DNA sequence. Residue numbering in theproteins encoded by the cDNA inserts are as in the Swiss-Prot data bank.Deletion mutants were produced by PCR, and point mutants byoligonucleotide-directed mutagenesis (Current Protocols in Molec. Biol.,1994).

(ii) Induced Expression, Metabolic Labeling and Immunoprecipitation ofProteins

MORT-1, N-linked to the FLAG octopeptide (FLAG-HF1; Eastman Kodak, NewHaven, Ct., USA), Fas-IC, FAS-R, p55-R′ a chimera comprised of theextracellular domain of p55-R (amino acids 1-168) fused to thetransmembrane and intracellular domain of FAS-R (amino acids 153-319),and the luciferase cDNA which serves as a control, were expressed inHeLa cells. Expression was carried out using a tetracycline-controlledexpression vector, in a HeLa cell clone (HtTA-I) that expresses atetracycline-controlled transactivator (Gossen and Bujard, 1992; asdescribed in PCT/US95/05854; see also Boldin et al., 1995). Metaboliclabeling with [³⁵S]methionine and [³⁵S]cysteine (DuPont, Wilmington,Del., USA and Arnersham, Buckinghamshire, England) was performed 18hours after transfection, by a further 4-hour incubation at 37° C. inDulbecco's modified Eagle's medium lacking methionine and cysteine, butsupplemented with 2% dialyzed fetal calf serum. The cells were thenlysed in RIPA buffer (10 mM Tris-HCI, pH 7.5, 150 mM NaCl, 1% NP-40, 1%deoxycholate, 0.1% SDS and 1 mM EDTA) and the lysate was pre-cleared byincubation with irrelevant rabbit antiserum (3 μl/ml) and Protein GSepharose beads (Pharmacia, Uppsala, Sweden; 60 μl/ml).Immunoprecipitation was performed by 1-hour incubation at 4° C. of 0.3ml aliquots of lysate with mouse monoclonal antibodies (5 μl/aliquot)against the FLAG octopeptide (M2; Eastman Kodak), p55-R (#18 and #20;(Engelmann et al., 1990)), or FAS-R (ZB4; Kamiya Southand Oaks, Calif.,USA), or with isotype matched mouse antibodies as a control, followed bya further 1-hour incubation with Protein G Sepharose beads (30μl/aliquot).

(iii) In Vitro Binding

Glutathione S-transferase (GST) fusions with the wild type or a mutatedFAS-IC were produced and adsorbed to glutathione-agarose beads (asdescribed in L 109632, 111125, 112002, 112692; see also Boldin et al.,1995; Current Protocols in Molecular Biology, 1994; Frangioni and Neel,1993). Binding of metabolically-labeled FLAG-HF1 fusion protein toGST-Fas-IC was assessed by incubating the beads for 2 hours at 4° C.with extracts of HeLa cells, metabolically labeled with [³⁵S]methionine(60 μCi/ml), that express FLAG-HF1. The extracts were prepared in abuffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mMdithiotreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 μg/mlAprotonin, 20 μg/ml Leupeptin, 10 mM sodium fluoride and 0.1 mM sodiumvanadate (1 ml per 5×10⁵ cells).

(iv) Assessment of the Cytotoxicity Triggered by Induced Expression ofHF1

HF1, Fas-IC, p55-IC and luciferase cDNAs were inserted into atetracycline-controlled expression vector and transfected to HtTA-1cells (a HeLa cell line) (Gossen and Bujard, 1992) together with thesecreted placental alkaline phosphatase cDNA, placed under control ofSV40 promoter (the PSBC-2 vector (Dirks et al., 1993)). Cell death wasassessed 40 hours after transfection, either by the neutral-red uptakeassay (Wallach, 1984) or, for assessing specifically the death in thosecells that express the transfected cDNAs, by determining the amounts ofplacental alkaline phosphatase (Berger et al., 1988) secreted to thegrowth medium at the last 5 hours of incubation.

In another set of experiments to analyze the region of the MORT-1 (HF1)protein involved in the binding to the FAS-IC, the following proteinswere expressed transiently in HeLa cells that contain atetracycline-controlled transactivator (HtTA-1), using atetracycline-controlled expression vector (pUHD10-3): Human FAS-R alone;Human FAS-R as well as the N-terminal part of MORT-1 (amino acids 1-117,the “MORT-1 head”); Human FAS-R as well as the C-terminal part ofMORT-1, which contains its ‘death domain’ homology region (amino acids130-245, the “MORT-1 DD”, see also L 112742); FLAG-55.11 (amino acids309-900 of protein 55.11 fused at the N-terminus to the FLAGoctapeptide, the protein 55.11 being a p55-IC-specific binding protein,see also L 109632). Twelve hours after transfection, the cells weretrypsinized and re-seeded at a concentration of 30,000 cells/well. After24 hours further incubation, the cells were treated for 6 hours with amonoclonal antibody against the extracellular domain of FAS-R(monoclonal antibody CH-11, Oncor) at various concentrations (0.001-10μg/ml monoclonal antibody), in the presence of 10 g/ml cycloheximide.Cell viability was then determined by the neutral-red uptake assay andthe results were presented in terms of % viable cells as compared tocells that had been incubated with cycloheximide alone (in the absenceof anti-FAS-R monoclonal antibody CH11).

(v) Northern and Sequence Analyses

poly A⁺RNA was isolated from total RNA of HeLa cells (Oligotex-dT mRNAkit. QIAGEN, Hilden, Germany). Northern analysis using the HF1 cDNA as aprobe was performed by conventional methods (as described inPCT/US95105854; see also Boldin et al., 1995). The nucleotide sequenceof MORT-1 (HF1) was determined in both directions by the dideoxy chaintermination method.

The results (shown in Table 1 and FIGS. 1-7) obtained from the aboveexperimental procedures are as follows: Sequence analysis of the MORT-1cDNA cloned by the two-hybrid procedure indicated that it encodes anovel protein 5 (see below). Applying the two-hybrid test further toevaluate the specificity of the binding of this protein (MORT-1) for“Mediator of Receptor-induced Toxicity”) to FAS-IC, and to define theparticular region in FAS-IC to which it binds, led to the followingfindings (Table 1): (a) The protein binds =0 both to human and to mouseFAS-IC, but not to several other tested proteins, including threereceptors of the TNF/NGF receptor family (p55 and p75 TNF receptors andCD40); (b) Replacement mutations at position 225 (Ile) in the ‘deathDomain’ of FAS-R, shown to abolish signaling both in vitro and in vivo(the lpr^(cg) mutation (Watanabe-Fukunaga et al., 1992; Itoh and Nagata,1993), also prevents binding of MORT-1 to the FAS-IC; (c) The MORT-1binding-site in FAS-R occurs within the ‘death domain’ of this receptor;and (d) MORT-1 binds to itself. This self-binding, and the binding ofHF1 to FAS-R involve different regions of the protein: A fragment ofMORT-1 corresponding to residues 1-117 binds to the full-length MORT-1,but does not bind to itself nor to the FAS-IC. Conversely, a fragmentcorresponding to residues 130-245 binds to FAS-R, yet does not bind toMORT-1 (Table 1). Furthermore, it is apparent from the results in Table1 that the ‘death domain’ region of FAS-R is critical for FAS-ICself-association, as is the ‘death domain’ region of p55-R for p55-ICself-association. The deletions on both sides of these ‘death domains’does not affect the self-association ability thereof while, however, adeletion within these ‘death domains’ does affect the self-association.In the case of MORT-1, the binding of MORT-1 to FAS-IC is also dependentupon the complete (full) ‘death domain’ of FAS-R, while however, it isalso not dependent on the regions outside of the FAS-R ‘death domain’region for FAS-IC binding.

TABLE 1 Interaction of MORT-1 with FAS-IC and Self-Association of MORT-1within Transformed Yeasts: Assessment by a Two-Hybrid β-GalactoseExpression Test ACTIVATION-DOMAIN HYBRID DNA-BINDING - MORT1 humanDOMAIN HYBRID Full 1-117 130-245 Fas-IC SNF4 pGAD-GH Human Fas-IC

Full ++ − + ++ − −

200-319 ++ ++ − −

233-319 − − − −

175-304 ++ − + ++ − − Mouse Fas-IC

Full ++ − ++ ++ − −

197-306 ++ − + ++ − −

I225N − − − ++ − −

I225A − ++ − − MORT1

Full ++ ++ − ++ − −

1-117 + − − − −

130-245 − − ++ − − SPECIFICITY TESTS human p55-IC − − − − human p75-IC −− − − human CD40-IC − − − − Cyclin D − − − − Lamin − − − − SNF1 − − + −pGBT9 − − − −

In Table 1 above there is depicted the interaction of the proteinsencoded by the Gal4 DNA binding domain and activation-domain constructs(pGBT9 and pGAD-GH) within transfected SFY526 yeasts as assessed byβ-galactosidase expression filter assay. The DNA-binding-domainconstructs included four constructs of the human FAS-IC, four constructsof the mouse FAS-IC including two full-length constructs having Ile toLeu or Ile to Ala replacement mutations at position 225 (I225N and1225A, respectively), and three HF1 (MORT-1) constructs, all of whichconstructs are shown schematically on the left-hand side of the table.The activation-domain constructs included three HF1 constructs, the HF1portion being as in the DNA-binding-domain constructs; and a full-lengthhuman FAS-IC construct, the FAS-IC portion being the same as in theabove DNA-binding domain construct. The intracellular domains of humanp55 TNF receptor (p55-IC residues 206-426), human CD40 (CD40-IC,residues 216-277) and human p75 TNF receptor (p75-IC, residues 287-461)as well as lamin, cyclin D and ‘empty’ Gal4 (pGBT9) vectors served asnegative controls in the form of DNA-binding domain constructs. SNF1 andSNF4 served as positive controls in the form of DNA-binding-domain(SNF1) and activation-domain (SNF4) constructs. ‘Empty’ Gal4 vectors(pGAD-GH) also served as negative controls in the form of activationdomain constructs (for more details concerning the p55-IC, p75-IC, seealso PCT/US95/05854). The symbols “++” and “+” denote the development ofstrong color within 30 and 90 minutes of the assay, respectively; and“-” denotes no development of color within 24 hours. Combinations forwhich no score is given have not been tested.

Expression of HF1 (MORT-1) molecules fused at their N terminus with theFLAG octapeptide (FLAG-HF1) yielded in HeLa cells proteins of fourdistinct sizes—about 27, 28, 32, and 34 kD. In FIGS. 1 (A and B) thereis shown the results demonstrating the interaction of HF1 with FAS-IC invitro. As noted above in the description of FIGS. 1A and B, FIG. 1A is areproduction of a control autoradiogram of an immunoprecipitate ofproteins from extracts of HeLa cells transfected with the FLAG-HF1(FLAG-MORT1) fusion protein or with luciferase cDNA as a control, theimmunoprecipitation being performed with anti-FLAG antibody (αFLAG).FIG. 1B is a reproduction of an autoradiogram showing the interaction invitro between HF1 and FAS-IC wherein the HF1 is in the form of[³⁵S]methionine-metabolically labeled HF1-FLAG fusion proteins obtainedfrom extracted of transfected HeLa cells and the FAS-IC is in the formof human and mouse GST-FAS-IC fusion proteins including one having areplacement mutation at position 225 in FAS-IC, all of which GST-FAS-ICfusion proteins were produced in E. coli. The GST-fusion proteins wereattached to glutathione beads before interaction with the extractscontaining the HF1-FLAG fusion protein following this interaction,SDS-PAGE was performed. Thus the in vitro interaction was evaluated byassessing, by autoradiography following SDS-PAGE, the binding of [³⁵S]metabolically labeled HF1, produced in transfected HeLa cells as afusion with the FLAG octapeptide (FLAG-HF1), to GST, GST fusion with thehuman or mouse FAS-IC (GST-huFAS-IC, GST-mFAS-IC) or to GST fusion withFAS-IC containing an Ile to Ala replacement mutation at position 225. Asis apparent from FIG. 1B, all four FLAG-HF1 proteins showed ability tobind to FAS-IC upon incubation with a GST-FAS-IC fusion protein. As inthe yeast two-hybrid test (Table 1), HF1 did not bind to a GST-FAS-ICfusion protein with a replacement at the lpr^(cg) mutation site (1225A).

The proteins encoded by the FLAG-HF1 cDNA showed also an ability to bindto the intracellular domain of FAS-R, as well as to the intracellulardomain of FAS-R chimera whose extracellular domain was replaced withthat of p55-R (p55-FAS), when co-expressed with these receptors in HeLacells. In FIGS. 2 (A, B, C) there is shown the results demonstrating theinteraction of HF1 with FAS-IC in transfected HeLa cells, i.e., in vivo.As mentioned above in the description of FIGS. 2A, B, C, these figuresare reproductions of autoradiograms of immunoprecipitates of varioustransfected HeLa cells which demonstrate the in vivo interaction andspecificity of the interaction between HF1 and FAS-IC in cellsco-transfected with constructs encoding these proteins. Thus, FLAG-HF1fusion protein was expressed and metabolically labeled with [35S]cysteine (20 μCi/ml) and [³⁵S]methionine (40 μCi/ml) in HeLa cells,alone, or together with human FAS-R, FAS-R chimera in which theextracellular domain of FAS-R was replaced with the corresponding regionin the human p55-R (p55-FAS), or the human p55-R, as negative control.Cross-immunoprecipitation of HF1 with the co-expressed receptor wasperformed using the indicated antibodies (FIGS. 2AC). As is apparent inFIGS. 2A-C, FLAG-HF1 is capable of binding to the intracellular domainof FAS-R, as well as to the intracellular domain of a FAS-R-p55-Rchimera having the extracellular domain of p55-R and the intracellulardomain of FAS-R, when co-expressed with these receptors in the HeLacells (see 3 middle lanes FIGS. 2A and 3 left-hand lanes FIG. 2C,respectively). Further, immunoprecipitation of FLAG-HF1 from extracts ofthe transfected cells also resulted in precipitation of the co-expressedFAS-R (FIG. 2A) or the co-expressed p55-FAS chimera (FIG. 2C).Conversely, immunoprecipitation of these receptors resulted in theco-precipitation of the FLAG-HF1 (FIGS. 2A and 2C).

Northern analysis using the HF1 cDNA as probe revealed a singlehybridizing transcript in HeLa cells. FIG. 3 is shows a reproduction ofa Northern blot in which poly A⁺ RNA (0.3 μg) from transfected cells washybridized with the HF1 cDNA. The size of this transcript (about 1.8 kB)is close to that of the HF1 cDNA (about 1702 nucleotides).

In sequence analysis, the cDNA was found to contain an open readingframe of about 250 amino acids. FIG. 4 depicts the preliminarynucleotide and deduced amino acid sequence of HF1 in which the ‘deathdomain’ motif is underlined, as is a possible start Met residue(position 49; bold, underlined M) and the translation stop codon (theasterisk under the codon at position 769-771). This ‘death domain’ motifshares homology with the known p55R and FAS-R ‘death domain’ motifs(p55-DD and FAS-DD). In order to determine the precise C-terminal end ofHF1 and to obtain evidence concerning the precise N-terminal (initialMet residue) end of HF1, additional experiments were carried out asfollows:

Using the methods described above, a number of constructs encoding HF1molecules fused at their N-terminus with the FLAG octapeptide (FLAG-HF1)were constructed and expressed in HeLa cells with metabolic labeling ofthe expressed proteins using 35S-cysteine and ³⁵S-methionine (see theabove in respect of FIG. 5B). The HF1-FIAG molecules were encoded by thefollowing cDNAs containing different portions of the HF1-encodingsequence:

(i) The FLAG octapeptide cDNA linked to the 5′ end of the HF1 cDNA fromwhich nucleotides 1-145 (see FIG. 4) have been deleted;

(ii) The FLAG octapeptide cDNA linked to the 5′ end of the HF1full-length cDNA (see FLAG-HF1 construct above in respect of FIG. 1B);

(iii) The FLAG octapeptide cDNA linked to the 5′ end of the HF1 cDNAfrom which nucleotides 1-145 as well as nucleotides 832-1701 (see FIG.4) had been deleted and the codon GCC at position 142-144 was mutated toTCC to prevent start of translation at this site.

Following expression of the above HF1-FLAG fusion products,immunoprecipitation was carried out as mentioned above, using eitheranti-FLAG monoclonal antibodies (M2) or as a control, anti-p75 TNF-Rantibodies (#9), followed by SDS-PAGE (10% acrylamide) andautoradiography. The results are shown in FIG. 5 which is a reproductionof an autoradiogram on which was separated the above noted HF1-FLAGfusion proteins, the samples loaded on each lane of the gel being asfollows:

Lanes 1 and 2: HF1-FLAG fusion protein encoded by the FLAG octapeptidecDNA linked to the 5′ end of the HF1 cDNA from which nucleotides 1-145had been deleted.

Lanes 3 and 4: HF1-FLAG fusion protein encoded by the FLAG octapeptidecDNA linked to the 5′ end of the full length HF1 cDNA.

Lanes 5 and 6: HF-1-FLAG fusion protein encoded by the FLAG octapeptidecDNA linked to the 5′ end of the HF1 cDNA from which nucleotides 1-145as well as 832-1701 had been deleted and the GCC at position 142-144 wasmutated to TCC to prevent start of translation at this site.

The immunoprecipitations were with anti-FLAG monoclonal antibodies forthe samples in lanes 2, 4 and 6 and anti-p75 TNF-R antibodies for thesamples in lanes 1, 3 and 5.

From the autoradiogram of FIG. 5 it is apparent that the identity ofproduct sizes in lanes 2 and 4 confirms that the nucleotides 769-771 arethe site of translation termination for HF1, i.e., this codon representsa stop signal and is indicated by an asterisk in FIG. 4. Further, theoccurrence of a broad band which represents just two translationproducts (as seen on the gel but being strongly labeled becomes a singlelarge band on the autoradiogram) in lane 6 indicates that the occurrenceof two additional products (the higher molecular weight broad bands) inlanes 2 and 4 reflect the initiation of translation both at theN-terminus of the FLAG-HF1 fusion molecule and at the methionine residuenumber 49 within the HF1 sequence (see bold underlined M at position 49of the amino acid sequence in FIG. 4). Thus, the above results haveconfirmed (validated) the C-terminal end of HF1 and have providedevidence that the N-terminal end of HF 1 may be at position 49 of thesequence in FIG. 4.

Indeed, it has been shown by additional expression experiments of HF1without the FLAG octapeptide fused to its 5-end, that Met⁴⁹ serves as aneffective site of translation initiation.

It should be mentioned that a search conducted in the ‘Gene Bank’ andProtein Bank’ DataBases revealed that there is no sequence correspondingto that of HF1 depicted in FIG. 4. Thus, HF1 represents a newFAS-IC-specific binding protein.

High expression of p55-IC results in triggering of a cytocidal effect(see L 109632, 111125 and Boldin et al., 1995). The expression of FAS-ICin HeLa cells also has such an effect, though to a lower extent, whichcould be detected only with the use of a sensitive assay. In FIGS. 6Aand B there is depicted graphically the ligand independent triggering ofcytocidal effects in cells transfected with HF1, as well as human p55-ICand FAS-IC. The effect of transient expression of HF1, human FAS-IC,human p55-IC, or luciferase that served as a control, on the viabilityof HeLa cells was assessed using a tetracycline-controlled expressionvector. Cell viability was evaluated 40 minutes after transfecting thesecDNAs either in presence (open bars, FIGS. 6A and B) or absence (closedbars, FIGS. 6A and B) of tetracycline (1 μg/ml, to block expression),together with a cDNA encoding the secreted placental alkalinephosphatase. Cell viability was determined either by the neutral reduptake assay (FIG. 6A) or, for determining specifically the viability ofthose particular cells that express the transfected DNA, by measuringthe amounts of placental alkaline phosphatase secreted to the growthmedium (FIG. 6B).

Thus, it is apparent from FIGS. 6A and B that the expression of HF1 inHeLa cells resulted in significant cell death, greater than that causedby FAS-IC expression. These cytotoxic effects of all of p55-IC, FAS-ICand HF1 seem to be related to the ‘death domain’ regions, present in allof these proteins, which ‘death domains’ have a propensity toself-associate, and thereby possibly prompting the cytotoxic effects.

In view of the above mentioned characteristics of HF1 (MORT-1), namely,the specific association of HF1 with that particular region in FAS-Rwhich is involved in cell death induction, and the fact that even aslight change of structure in that region, which prevents signaling (thelpr^(cg) mutation) abolishes also the binding of HF1, indicates thatthis protein plays a role in the signaling or triggering of cell death.This notion is further supported by the observed ability of HF1 totrigger by itself a cytocidal effect. Thus, HF1 (MORT-1) may function as(i) a modulator of the self-association of FAS-R by its own ability tobind to FAS-R as well as to itself, or (ii) serve as a docking site foradditional proteins that are involved in the FAS-R signaling, i.e., HF1may be a ‘docking’ protein and may therefore bind other receptorsbesides FAS-R, or (iii) constitutes part of a distinct signaling systemthat interacts with FAS-R signaling.

In order to further analyze the regions of MORT-1 (HF1) involved inFAS-IC binding and modulation of the FAS-R-mediated cellular effects(cytotoxicity), the abovementioned experiments were carried out, usingvectors encoding portions of MORT-1 (the ‘MORT-1 head’, amino acids10117 and the ‘MORT-1 dd’, amino acids 130-245) (separately), with avector encoding the human FAS-R for co-transfections of HeLa cells. Inthese experiments the various proteins and combinations of proteins wereexpressed transiently in HeLa cells that contain atetracycline-controlled transactivator (HtTA-1) by inserting thesequences encoding the proteins into a tetracycline-controlledexpression vector PUHD10-3. Control transfections employed vectorsencoding only the FAS-R and vectors encoding the FLAG-55.11 fusionprotein (the 55.11 protein being a p55-IC-specific binding protein ofwhich a portion containing amino acids 309-900 was fused (at itsN-terminal) to the FLAG octapeptide).

Following the transfection and incubation periods (see (iv) above) thetransfected cells were treated with various concentrations of ananti-FAS-R monoclonal antibody (CH-11) which binds specifically to theextracellular domain of FAS-R expressed by cells. This binding ofanti-FAS-R antibody induces the aggregation of the FAS-R at the cellsurface (much like the FAS-R ligand) and induces the intracellularsignaling pathway mediated by the FAS-IC, resulting, ultimately, in celldeath (FAS-R mediated cell cytotoxicity). The concentrations of theanti-FAS-R monoclonal antibody (CH11) used were in the range of 0.01-10μg/ml, usually concentrations such as 0.005; 0.05; 0.5 and 5 μg/ml. Thecells were treated with the anti-FAS antibody in the presence of 10μg/ml cycloheximide.

The results of the above analysis are set forth graphically in FIG. 7which depicts the % viability of the transfected cells as a function ofthe concentration of anti-FAS-R monoclonal antibody (CH11) used totreated the ceils, for each of the four different groups of transfectedcells. These groups of transfected cells are denoted by differentsymbols as follows: (i) the open squared denote cells transfected onlywith the non-relevant (i.e., non-FAS-IC binding) control vector encodingthe FLAG-55.11 fusion protein (“55.11”, negative control); (ii) theclosed squared denote cells co-transfected with vectors encoding theFAS-R and vectors encoding the C-terminal portion of MORT-1, amino acids130-245, which contains the MORT-1 ‘death domain’ (dd) homology region(“fas+mortldd”); (iii) the closed triangles denote cells transfectedwith only the vector encoding the FAS-R (“fas”, positive control); and(iv) the open circles denote cells co-transfected with vectors encodingthe FAS-R and vectors encoding the N-terminal portion of MORT-1, aminoacids 1-117, the ‘MORT1 head’ (“fas+mortlhe”).

From the results presented in FIG. 7, it is apparent that the expressionof FAS-R in the transfected cells conveys an increased sensitivity tothe cytocidal effects of the anti-FAS-R antibodies (compare “fas” to“55.11”). Further, the co-expression of the region in MORT1 thatcontains the ‘death domain’homology region and FAS-R (“fas+mortldd”)strongly interferes with FAS-induced (i.e., FAS-R mediated) cell deathas would be expected from the ability (see Table 1 above) of the MORT-1‘death domain’ (DD) region to bind to the FAS-R ‘death domain’ (FAS-DD).Moreover, co-expression of the N-terminal part of MORT-1 and FAS-R(“fas+mortlhe”) does not interfere with FAS-R-mediated cell death and,if at all, somewhat enhances the cytotoxicity (i.e., slightly increasedcell death)

Thus, the above results clearly indicate that the MORT-1 protein has twodistinct regions as far as binding to the FAS-IC and mediation of thecell-cytotoxic activity of the FAS-IC are concerned.

These results therefore also provide a basis for the use of differentparts (i.e., active fragments or analogs) of the MORT-1 protein fordifferent pharmaceutical applications. For example, the analogs orfragments or derivatives thereof of the MORT-1 protein which containessentially only the C-terminal portion of MORT-1 inclusive of its‘death domain’ region may be used for inhibiting FAS-R-mediatedcytotoxic effects in FAS-R containing cells or tissues and therebyprotect these cells or tissues from the deleterious effects of the FAS-Rligand in cases such as, for example, acute hepatitis. Alternatively,the analogs or fragments or derivatives thereof of the MORT-1 proteinwhich contain essentially only the N-terminal portion of MORT-1 may beused for enhancing the FAS-R-mediated cytotoxic effects in FAS-Rcontaining cells and tissues, thereby leading to the enhanceddestruction of these cells or tissues when desired in cases such as, forexample, tumor cells and autoreactive T and B cells. As detailed hereinabove, the above uses of the different regions of MORT-1 may be carriedout using the various recombinant viruses (e.g., Vaccinia) to insert theMORT-1 region-encoding sequence into specific cells or tissues it isdesired to treat.

Furthermore, it is also possible to prepare and use various othermolecules such as, antibodies, peptides and organic molecules which havesequences or molecular structures corresponding to the above notedMORT-1 regions in order to achieve the same desired effects mediated bythese MORT-1 regions.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 1701<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(768) <400> SEQUENCE: 1gtg aat cag gca ccg gag tgc agg ttc ggg gg#t gga atc ctt ggg ccg       48Val Asn Gln Ala Pro Glu Cys Arg Phe Gly Gl #y Gly Ile Leu Gly Pro1               5    #                10   #                15ctg ggc aag cgg cga gac ctg gcc agg gcc ag#c gag ccg agg aca gag       96Leu Gly Lys Arg Arg Asp Leu Ala Arg Ala Se #r Glu Pro Arg Thr Glu            20       #            25       #            30ggc gcg cgg agg gcc ggg ccg cag ccc cgg cc#g ctt gca gac ccc gcc      144Gly Ala Arg Arg Ala Gly Pro Gln Pro Arg Pr #o Leu Ala Asp Pro Ala        35           #        40           #        45atg gac ccg ttc ctg gtg ctg ctg cac tcg gt#g tcg tcc agc ctg tcg      192Met Asp Pro Phe Leu Val Leu Leu His Ser Va #l Ser Ser Ser Leu Ser    50               #    55               #    60agc agc gag ctg acc gag ctc aag ttc cta tg#c ctc ggg cgc gtg gtc      240Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cy #s Leu Gly Arg Val Val65                   #70                   #75                   #80aag cgc aag ctg gag cgc gtg cag agc ggc ct#a gac ctc ttc tcc atg      288Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Le #u Asp Leu Phe Ser Met                85   #                90   #                95ctg ctg gag cag aac gac ctg gag ccc ggg ca#c acc gag ctc ctg cgc      336Leu Leu Glu Gln Asn Asp Leu Glu Pro Gly Hi #s Thr Glu Leu Leu Arg            100       #           105       #           110gag ctg ctc gcc tcc ctg cgg cgc cac gac ct#g ctg cgg cgc gtc gac      384Glu Leu Leu Ala Ser Leu Arg Arg His Asp Le #u Leu Arg Arg Val Asp        115           #       120           #       125gac ttc gag gcg ggg gcg gcg gcc ggg gcc gc#g cct ggg gaa gaa gac      432Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Al #a Pro Gly Glu Glu Asp    130               #   135               #   140ctg tgt gca gca ttt aac gtc ata tgt gat aa#t gtg ggg aaa gat tgg      480Leu Cys Ala Ala Phe Asn Val Ile Cys Asp As #n Val Gly Lys Asp Trp145                 1 #50                 1 #55                 1 #60aga agg ctg gct cgt cag ctc aaa gtc tca ga#c acc aag atc gac agc      528Arg Arg Leu Ala Arg Gln Leu Lys Val Ser As #p Thr Lys Ile Asp Ser                165   #               170   #               175atc gag gac aga tac ccc cgc aac ctg aca ga#g cgt gtg cgg gag tca      576Ile Glu Asp Arg Tyr Pro Arg Asn Leu Thr Gl #u Arg Val Arg Glu Ser            180       #           185       #           190ctg aga atc tgg aag aac aca gag aag gag aa#c gca aca gtg gcc cac      624Leu Arg Ile Trp Lys Asn Thr Glu Lys Glu As #n Ala Thr Val Ala His        195           #       200           #       205ctg gtg ggg gct ctc agg tcc tgc cag atg aa#c ctg gtg gct gac ctg      672Leu Val Gly Ala Leu Arg Ser Cys Gln Met As #n Leu Val Ala Asp Leu    210               #   215               #   220gta caa gag gtt cag cag gcc cgt gac ctc ca#g aac agg agt ggg gcc      720Val Gln Glu Val Gln Gln Ala Arg Asp Leu Gl #n Asn Arg Ser Gly Ala225                 2 #30                 2 #35                 2 #40atg tcc ccg atg tca tgg aac tca gac gca tc#t acc tcc gaa gcg tcc      768Met Ser Pro Met Ser Trp Asn Ser Asp Ala Se #r Thr Ser Glu Ala Ser                245   #               250   #               255tgatgggccg ctgctttgcg ctggtggacc acaggcatct acacagcctg ga#ctttggtt    828ctctccagga aggtagccca gcactgtgaa gacccagcag gaagccaggc tg#agtgagcc    888acagaccacc tgcttctgaa ctcaagctgc gtttattaat gcctctcccg ca#ccaggccg    948ggcttgggcc ctgcacagat atttccattt cttcctcact atgacactga gc#aagatctt   1008gtctccacta aatgagctcc tgcgggagta gttggaaagt tggaaccgtg tc#cagcacag   1068aaggaatctg tgcagatgag cagtcacact gttactccac agcggaggag ac#cagctcag   1128aggcccagga atcggagcga agcagagagg tggagaactg ggatttgaac cc#ccgccatc   1188cttcaccaga gcccatgctc aaccactgtg gcgttctgct gcccctgcag tt#ggcagaaa   1248ggatgttttt gtcccatttc cttggaggcc accgggacag acctggacac ta#gggtcagg   1308cggggtgctg tggtggggag aggcatggct ggggtggggg tggggagacc tg#gttggccg   1368tggtccagct cttggcccct gtgtgagttg agtctcctct ctgagactgc ta#agtagggg   1428cagtgatggt tgccaggacg aattgagata atatctgtga ggtgctgatg ag#tgattgac   1488acacagcact ctctaaatct tccttgtgag gattatgggt cctgcaattc ta#cagtttct   1548tactgttttg tatcaaaatc actatctttc tgataacaga attgccaagg ca#gcgggatc   1608tcgtatcttt aaaaagcagt cctcttattc ctaaggtaat cctattaaaa ca#cagcttta   1668 caacttccat attacaaaaa aaaaaaaaaa aaa       #                   #       1701 <210> SEQ ID NO 2 <211> LENGTH: 256<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Val Asn Gln Ala Pro Glu Cys Arg Phe Gly Gl #y Gly Ile Leu Gly Pro1               5    #                10   #                15Leu Gly Lys Arg Arg Asp Leu Ala Arg Ala Se #r Glu Pro Arg Thr Glu            20       #            25       #            30Gly Ala Arg Arg Ala Gly Pro Gln Pro Arg Pr #o Leu Ala Asp Pro Ala        35           #        40           #        45Met Asp Pro Phe Leu Val Leu Leu His Ser Va #l Ser Ser Ser Leu Ser    50               #    55               #    60Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cy #s Leu Gly Arg Val Val65                   #70                   #75                   #80Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Le #u Asp Leu Phe Ser Met                85   #                90   #                95Leu Leu Glu Gln Asn Asp Leu Glu Pro Gly Hi #s Thr Glu Leu Leu Arg            100       #           105       #           110Glu Leu Leu Ala Ser Leu Arg Arg His Asp Le #u Leu Arg Arg Val Asp        115           #       120           #       125Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Al #a Pro Gly Glu Glu Asp    130               #   135               #   140Leu Cys Ala Ala Phe Asn Val Ile Cys Asp As #n Val Gly Lys Asp Trp145                 1 #50                 1 #55                 1 #60Arg Arg Leu Ala Arg Gln Leu Lys Val Ser As #p Thr Lys Ile Asp Ser                165   #               170   #               175Ile Glu Asp Arg Tyr Pro Arg Asn Leu Thr Gl #u Arg Val Arg Glu Ser            180       #           185       #           190Leu Arg Ile Trp Lys Asn Thr Glu Lys Glu As #n Ala Thr Val Ala His        195           #       200           #       205Leu Val Gly Ala Leu Arg Ser Cys Gln Met As #n Leu Val Ala Asp Leu    210               #   215               #   220Val Gln Glu Val Gln Gln Ala Arg Asp Leu Gl #n Asn Arg Ser Gly Ala225                 2 #30                 2 #35                 2 #40Met Ser Pro Met Ser Trp Asn Ser Asp Ala Se #r Thr Ser Glu Ala Ser                245   #               250   #               255

What is claimed is:
 1. A method for isolating and identifyingpolypeptides capable of binding to a MORT-1 polypeptide comprises:bringing a polypeptide to be screened into contact with said MORT-1polypeptide and producing any polypeptide identified as being capable ofbinding to MORT-1, wherein said MORT-1 polypeptide comprises: (1) theMORT-1 protein having the amino acid sequence of SEQ ID NO:2; (2) ananalog of said MORT-1 protein which differs therefrom by a single aminoacid residue and binds with the intracellular domain of the FAS ligandreceptor (FAS-IC); or (3) a fragment of said MORT-1 protein which bindswith FAS-IC.
 2. A method according to claim 1, wherein said MORT-1polypeptide comprises the MORT-1 protein having the amino acid sequenceof SEQ ID NO:2.
 3. A method according to claim 1, further comprisingapplying the procedure of affinity chromatography in which said MORT-1polypeptide is attached to an affinity chromatography matrix, whereinsaid MORT-1 polypeptide is brought into contact with a cell extract insaid bringing into contact step and polypeptides from the cell extractwhich binds to said attached MORT-1 are then eluted, isolated andanalyzed.
 4. A method according to claim 1, wherein said bring intocontact step comprises applying the yeast two-hybrid procedure in whicha sequence encoding said MORT-1 polypeptide is carried by one hybridvector and sequence from a cDNA or genomic DNA library is carried by thesecond hybrid vector, the vectors then being used to transform yeasthost cells and the positive transformed cells being isolated, followedby extraction of the said second hybrid vector to obtain a sequenceencoding a protein which binds to said MORT-1 polypeptide.
 5. A methodaccording to claim 3, wherein said MORT-1 polypeptide comprises theMORT-1 protein having the amino acid sequence of SEQ ID NO:2.
 6. Amethod according to claim 4, wherein said MORT-1 polypeptide comprisesthe MORT-1 protein having the amino acid sequence of SEQ ID NO:2.